The goal of this project continues to be the understanding of the role of viral-cellular interactions in the regulation of gene expression of the human immunodeficiency virus (HIV). We have studied the role of cis-acting DNA sequences in the HIV long terminal repeat (LTR) in the control of HIV gene expression in both acute infection of human T lymphocytes and in chronic or latent infections. In the past year, our studies have focused on the Sp1 binding sites in the HIV LTR. Virus containing a deletion of all three Sp1 sites replicates efficiently in PHA-stimulated human peripheral blood lymphocytes (PBLs) and in the MT4 human T cell line, but did not replicate in A3.01 cells unless those cells were treated with the cytokine, TNF-alpha. Gel retardation assays were performed to study the basis for the differential replication of the Sp1-deleted virus. While both MT4 cells and A3.01 cells contained abundant Sp1 binding activity, NF-kappaB activity could be detected in the nuclei of MT4 cells, but was not present in A3.01 cells unless those cells were treated with TNF-alpha. Thus, the presence of NF-kappaB binding activity in the nuclei of target T cells appeared to be required for the replication of proviruses deleted in the Sp1 sites. This suggests that the NTF-kappaB and Sp1 binding sites in the LTR share the properties of "enhansons" for HIV gene expression and may functionally substitute for each other in activating HIV replication. Recently we have identified revertants of the Sp1 deleted virus that have recovered the ability to replicate in A3.01 cells. These viruses retain the Sp1 site deletion but have an extra NF-kappaB binding site. The mechanisms by which a third NF-kappaB site now allows viral replication in A3.01 cells are currently under study.